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PubMed1977

Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells.

Ca2+-transport and its energy consumption were studied in intact human red cells loaded with Ca2+ by the aid of the ionophore A23187. After the complete elimination of the ionophore the passive Ca2+-permeability of the membrane returned to its normal low value, except when the intracellular Ca2+-concentration was higher than 3 mM or the ATP level fell below 100 muM. Within these limits the rate of Ca2+-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca2+]i and reached a plateau at about 1 mM intracellular Ca2+-concentration. The maximum rate of Ca2+-efflux was about 85 mumol/l of cells per min at 37 degrees C, pH 7.4. The activation energy of active Ca2+-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7. Ca2+-efflux was not connected with the counter-transport of cations. The Ca2+-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, N-ethylmaleimide and p-chloromercuribenzoate or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca2+-extrusion was brought about by 50-250 muM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca2+-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane. To examine the energy consumption of the Ca2+-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca2+-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na+-tetrathionate. Ca2+-activated ATPase activity connected with the Ca2+-pump was distinguished from other Ca2+-ATPases by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca2+-transported per ATP split was found to be 2 : 1.

Adenine Nucleotides↗
PubMed1983

Release of granule contents from sea urchin egg cortices. New assay procedures and inhibition by sulfhydryl-modifying reagents.

We have developed a rapid turbidimetric assay for the release of cortical granule contents from cortices prepared from eggs of the sea urchin, Strongylocentrotus purpuratus. The decrease in turbidity of cortex suspensions which occurs when the free calcium ion concentration is increased to 0.38-0.62 microM can be followed spectrophotometrically. Kinetic experiments demonstrate that this calcium-triggered turbidity change occurs rapidly and with no detectable lag period. Evidence indicating that the observed decrease in turbidity results from the release of cortical granule contents was obtained by correlating the free calcium ion concentration required to initiate the turbidity change with the free calcium ion concentration required in microscopic and enzymatic assays. All three assays exhibited similar calcium dependence. In the microscopic assay, morphological changes are used to assess the extent of cortical granule exocytosis. The enzymatic assay is based upon the latency of ovoperoxidase, a cortical granule enzyme. Ovoperoxidase catalyzed a 30-125-fold increase in the incorporation of [125I]iodine into trichloroacetic acid-precipitable cortex protein at and above threshold calcium ion concentrations. We have utilized the turbidimetric assay to screen several potential inhibitors of the cortical reaction. In confirmation of previous reports, we find that the phenothiazine drugs, chlorpromazine and trifluoperazine, are inhibitory. Sulfhydryl-modifying reagents, N-ethylmaleimide and sodium tetrathionate, are also inhibitory. Inhibition of cortical granule enzyme release by N-ethylmaleimide was confirmed with the ovoperoxidase latency assay.

Animals↗
PubMed1979

Effect of pH on eggshell penetration by Salmonellae.

Experiments were conducted to study effects of pH on penetration of eggs by three species of Salmonella. Eggs having an average specific gravity of 1.078 were subjected to challenge by either S. typhimurium, S. st. paul, or S. derby. Challenge solutions ranged from pH 5.0 to 9.5 in .5 pH increments and contained an average of 7.5 x 10(3) Salmonella/ml. Egg temperature was 22 C and solution temperature 4.4 C when challenged. Tartaric acid (10%) or 1 N. NaOH were used to adjust solution prior to adding challenge organisms. Eggs were challenged for 3 min then allowed to dry and held at 22 C for 24 hr, after which they were opened aseptically. Salmonella penetration was determined by swabbing the inner shell membrane and incubating in selenite cystine and tetrathionate enrichment broths for 24 hr followed by plating on MacConkey and SS agars. Penetration rates for all three organisms were significantly less at pH 5.0 than at any higher pH tested. There was an increase in penetration from pH 5.5 to 7.0 for all species. Maximum penetration rates were 42% of eggs challenged at pH 7.5, 22% at pH 8.5, and 34% at pH 7.0 for S. typhimurium, S. derby, and S. st. paul, respectively. In no case was penetration of eggs at pH 9.0 significantly different from pH at maximum penetration of challenge eggs. Penetration by S. st paul, at pH 9.5 was significantly less (P less than .05) than at pH 7.0. Decalcification of the eggshell was less than .01%/min at pH 4.0. Shell losses at pH 3.5 and 3.0 were .03% and .33%/min, respectively.

Animals↗
PubMed2003

Novel thermo-acidophilic bacteria isolated from geothermal sites in Yellowstone National Park: physiological and phylogenetic characteristics.

Moderately thermophilic acidophilic bacteria were isolated from geothermal (30-83 degrees C) acidic (pH 2.7-3.7) sites in Yellowstone National Park. The temperature maxima and pH minima of the isolates ranged from 50 to 65 degrees C, and pH 1.0-1.9. Eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrous iron in anaerobic cultures. Several of the isolates could also oxidize tetrathionate. Six of the iron-oxidizing isolates, and one obligate heterotroph, were low G+C gram-positive bacteria ( Firmicutes). The former included three Sulfobacillus-like isolates (two closely related to a previously isolated Yellowstone strain, and the third to a mesophilic bacterium isolated from Montserrat), while the other three appeared to belong to a different genus. The other two iron-oxidizers were an Actinobacterium (related to Acidimicrobium ferrooxidans) and a Methylobacterium-like isolate (a genus within the alpha -Proteobacteria that has not previously been found to contain either iron-oxidizers or acidophiles). The other three (heterotrophic) isolates were also alpha-Proteobacteria and appeared be a novel thermophilic Acidisphaera sp. An ARDREA protocol was developed to discriminate between the iron-oxidizing isolates. Digestion of amplified rRNA genes with two restriction enzymes ( SnaBI and BsaAI) separated these bacteria into five distinct groups; this result was confirmed by analysis of sequenced rRNA genes.

Bacteria↗
PubMed2001

Volatilization of mercury under acidic conditions from mercury-polluted soil by a mercury-resistant Acidithiobacillus ferrooxidans SUG 2-2.

Volatilization of mercury under acidic conditions from soil polluted with mercuric chloride (1.5 mg Hg/kg soil) was studied with resting cells of a mercury-resistant strain, Acidithiobacillus ferrooxidans SUG 2-2. When resting cells of SUG 2-2 (0.01 mg of protein) were incubated for 10 d at 30 degrees C in 20 ml of 1.6 mM sulfuric acid (pH 2.5) with ferrous sulfate (3%) and mercury-polluted soil (1 g), which contained 7.5 nmol of Hg, approximately 4.1 nmol of mercury was volatilized, indicating that 54% of the total mercury in the soil was volatilized. The amount of mercury volatilized from the soil was dependent on the concentration of Fe2+ added to the medium. When elemental sulfur, sodium tetrathionate, and pyrite were used as an electron donor for the mercury reduction, 16, 2.4 and 0.84%, respectively, of the total mercury added to the solution were volatilized. The optimum pH and temperature for mercury volatilization were 2.5 and 30 degrees C. Approximately 92% of the total mercury in a salt solution (pH 2.5) with resting cells of SUG 2-2 (0.01 mg of protein), ferrous sulfate (3%) and mercury-polluted soil (1 g) was volatilized by further addition of both resting cells and Fe2+ and by incubating for 30 d at 30 degrees C.

Hydrogen-Ion Concentration↗
PubMed1971

Comparison of media for isolation of salmonellae and shigellae from fecal specimens.

Five transport media, eight plating media, and three enrichment broth media for the isolation of salmonellae and shigellae were evaluated. Eight laboratories in widely separated regions of the United States participated in this evaluation by submitting 490 fecal specimens in the transport media provided. The results suggest that the newer transport media may not offer any advantage over the use of buffered glycerol-saline in the isolation of these enteric pathogens. Shigellae were best isolated by direct inoculation, whereas salmonellae were isolated in greater numbers after tetrathionate (without Brilliant Green) enrichment with subsequent culturing on the plating medium. The use of a variety of plating media is recommended for the recovery of a larger number of these enteric pathogens.

Agar↗
PubMedDate not supplied

[Two new species of microaerophilic sulfur spirilla, Spirillum winogradskii sp. nov. and Spirillum kriegii sp. nov].

New microaerophilic sulfur-oxidizing spirilla were isolated from hydrogen sulfide sludge of wastewater treatment plants. Strains D-427 and D-430 have spiral cells that are highly motile due to bipolar flagellum bundles covered with mucous sheaths. Under a phase-contrast microscope, these bundles are visible as single polar flagella. Spheroplasts are formed in the stationary growth phase. Both strains are obligate organotrophs able to oxidize a number of reduced sulfur compounds. The oxidation of sulfide and polysulfide leads to the formation of intracellular globules of elemental sulfur; thiosulfate oxidation results in tetrathionate accumulation in the medium. The cells are unable to utilize reduced sulfur compounds in the energy metabolism; their oxidation is caused by a chemical interaction with H2O2 and O2, synthesized in the electron transport chain. Both strains are obligate microaerophiles with an optimal oxygen concentration in the gas phase of 2 and 0.8% for strains D-427 and D-430, respectively. The strains utilize a limited number of organic acids as growth substrates, mainly tricarboxylic-acid-cycle intermediates. The DNA G+C content is 38.0 mol % (T(m)) for strain D-427 and 38.9 mol % for strain D-430. Phylogenetic analysis, based on the comparison of 16S rRNA gene sequences, revealed that the new isolates of sulfur spirilla are the most closely related to Spirillum volutans, the type species of the genus (97.4% similarity). They were assigned to the genus Spirillum within the class Beta-proteobacteria as two new species, S. winogradskii sp. nov. (D-427T = DSM 12756T) and S. kriegii sp. nov. (B-430T = BKM B-2372T). The emended description of the genus Spirillum is provided.

Genotype↗
PubMed2006

Synthesis and degradation test of hyaluronic acid hydrogels.

Hyaluronic acid (HA) hydrogels prepared with three different crosslinking reagents were assessed by in vitro and in vivo degradation tests for various tissue engineering applications. Adipic acid dihydrazide grafted HA (HA-ADH) was synthesized and used for the preparation of methacrylated HA (HA-MA) with methacrylic anhydride and thiolated HA (HA-SH) with Traut's reagent (imminothiolane). (1)H NMR analysis showed that the degrees of HA-ADH, HA-MA, and HA-SH modification were 69, 29, and 56 mol%, respectively. HA-ADH hydrogel was prepared by the crosslinking with bis(sulfosuccinimidyl) suberate (BS(3)), HA-MA hydrogel with dithiothreitol (DTT) by Michael addition, and HA-SH hydrogel with sodium tetrathionate by disulfide bond formation. According to in vitro degradation tests, HA-SH hydrogel was degraded very fast, compared to HA-ADH and HA-MA hydrogels. HA-ADH hydrogel was degraded slightly faster than HA-MA hydrogel. Based on these results, HA-MA hydrogels and HA-SH hydrogels were implanted in the back of SD rats and their degradation was assessed according to the pre-determined time schedule. As expected from the in vitro degradation test results, HA-SH hydrogel was in vivo degraded completely only in 2 weeks, whereas HA-MA hydrogels were degraded only partially even in 29 days. The degradation rate of HA hydrogels were thought to be controlled by changing the crosslinking reagents and the functional group of HA derivatives. In addition, the state of HA hydrogel was another factor in controlling the degradation rate. Dried HA hydrogel at 37 degrees C for a day resulted in relatively slow degradation compared to the bulk HA hydrogel. There was no adverse effect during the in vivo tests.

Adipates↗
PubMed2002

Specificity of respiratory pathways involved in the reduction of sulfur compounds by Salmonella enterica.

The tetrathionate (Ttr) and thiosulfate (Phs) reductases of Salmonella enterica LT2, together with the polysulfide reductase (Psr) of Wolinella succinogenes, are unusual examples of enzymes containing a molybdopterin active-site cofactor since all formally catalyse sulfur-sulfur bond cleavage. This is in contrast to the oxygen or hydrogen transfer reactions exhibited by other molybdopterin enzymes. Here the catalytic specificity of Ttr and Phs has been compared using both physiological and synthetic electron-donor systems. Ttr is shown to catalyse reduction of trithionate but not sulfur or thiosulfate. In contrast, Phs cannot reduce tetrathionate or trithionate but allows whole cells to utilize elemental sulfur as an electron acceptor. Mechanisms are proposed by which the bacterium is able to utilize an insoluble sulfur substrate by means of reactions at the cytoplasmic rather than the outer membrane.

Amino Acid Sequence↗
PubMed1986

Isolation of an amino terminal extended form of basic fibroblast growth factor.

Extraction of bovine pituitaries in the presence of enzyme inhibitors (2 mM PMSF, 2 mM sodium tetrathionate, 15 microM pepstatin A, and 1 mM EDTA) resulted in the isolation of two distinct forms of basic fibroblast growth factor. Partial characterization of both molecules showed one form to be identical to basic FGF(1-146) which has already been reported by our laboratory. The second form was estimated by SDS-PAGE to have a molecular weight of 17,000 Daltons which is slightly larger than that of basic FGF(1-146). Amino acid analysis shows the presence of 8 new residues more than basic FGF(1-146) which accounts for the difference in molecular weight. Gas-phase sequencing of this molecule indicated that it bears a blocked amino terminus. Furthermore, this higher molecular weight form of basic FGF did not show immunoreactivity with antibodies specific for the amino terminus of basic FGF(1-146) but cross reacted with antibodies generated against midportion fragments of basic FGF(1-146), indicating that the molecule is amino terminally extended. Like basic FGF(1-146), the molecule is a potent mitogenic factor for vascular endothelial cells. Taken together these results demonstrate the existence of a precursor form of basic FGF which is extended by 8 residues at the amino terminus with the first residue being blocked.

Amino Acids↗
PubMedDate not supplied

[Effect of cultivation conditions on the growth and activities of sulfur metabolism enzymes and carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41].

The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold-arsenic concentrate and elemental sulfur as a source of energy. The growth in the presence of S0 under auto- or mixotrophic conditions was less stable compared with the media containing iron monoxide. The enzymes involved in oxidation of sulfur inorganic compounds--thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodonase, adenylyl sulfate reductase, sulfite oxidase, and sulfur oxygenase--were discovered in the cells of Sulfobacillus grown in the mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle--ribulose bisphosphate carboxylase--and several other enzymes involved in heterotrophic fixation of carbonic acid. Activities of carboxylases depended on the composition of cultivation media.

Culture Media↗
PubMed1981

[Taxonomic study of enterobacteria belonging or related to Escherichia coli species].

Phenetic (numerical analysis) and genetic (DNA-DNA hybridization) studies were carried out on strains belonging or related to the species Escherichia coli. They have shown the diversity of its phenotypes, by the presence of plasmidic characters (citrate+, urease+, H2S+, tetrathionate reductase+, raffinose+, and saccharose+). New strains related phenetically to E. coli are also individualized. They showed less than 30% DNA relatedness with E. coli. A new definition of E. coli is presented.

Bacteriological Techniques↗
PubMed1990

Effects of various known and potential cyanide antagonists and a glutathione depletor on acute toxicity of cyanide in mice.

To compare the protective potencies of a large number of known and potential cyanide antagonists in one stock of mice, groups (N = 10) of male CF-1 Swiss-Webster mice were given a single maximal or near-maximal intraperitoneal injection of each substance. Ethyl maleate, a glutathione (GSH) depletor and potential enhancer of cyanide toxicity, was given to other groups. Thirty min later, the mice were given subcutaneous injections of graded doses of KCN. In untreated control mice, the 24-hr median lethal dose (LD50) of KCN was 11 mg/kg of body weight (potency ratio, PR = 1.0). In comparison, protective effects of traditional antagonists thiosulfate and nitrite produced PR values of 1.48 and 2.95, respectively. Tetrathionate, sulfate, dithionite, methionine, hydroxocobalamin, ascorbate, pyridoxal phosphate, alpha-ketoglutarate, alpha-ketobutyrate, GSH, GSH disulfide (GSSG) and selenite were similar in efficacy to thiosulfate (P less than 0.05; PR values 1.35-1.59). Cysteine, diethyldithiocarbamate (DEDC), and cobaltous chloride were more effective than thiosulfate (PR values 1.68, 1.69, and 1.85, respectively). Phentolamine and dicobalt EDTA were ineffective, whereas papaverine enhanced toxicity (PR 0.72). Agents with significant PR values (greater than or equal to 1.14) but which were less effective than thiosulfate included sulfite, dimercaptosuccinic acid, pyruvate, citrate, alpha-ketovalerate, naloxone, and corn oil. Ethyl maleate in corn oil markedly enhanced KCN lethality (PR 0.57 compared to corn oil alone), and caused prolonged illness in several mice. Vitamin E in corn oil had no effect. Dual mixtures of thiosulfate with other selected substances were also tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A potential anti-oxidant protein in a ferrous iron-oxidizing Sulfolobus species.

The production of a 25 kDa protein was greatly increased when a sulfur- and ferrous iron-oxidizing species of Sulfolobus was switched from growth on tetrathionate to growth on ferrous iron. The gene encoding the protein was cloned and sequenced. The predicted amino sequence showed significant similarity to those of the alkyl hydroperoxide reductase/thiol specific anti-oxidant family of proteins that appear to be involved in responses to certain types of oxidative stress.

Amino Acid Sequence↗
PubMed2003

Separation of thiosulfate and the polythionates in gold thiosulfate leach solutions by capillary electrophoresis.

A technique for the separation of thiosulfate (S(2)O(3) (2-)), polythionates (S(x)O(6) (2-), x = 3 to 5) and the gold(I) thiosulfate complex (Au(S(2)O(3))(2) (3-)) using capillary electrophoresis with simultaneous UV detection at 195 and 214 nm is presented. The five species were separated in under 3 min with a total analysis time of 8 min, using an electrolyte containing 25 mM 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol (bis-tris) adjusted to pH 6.0 with sulfuric acid and an applied voltage of -30 kV. While the gold(I) thiosulfate complex could be separated from the other analytes of interest under these conditions, the quantification of this complex was not possible due to inconsistent peak areas and peak splitting effects induced by the sulfur-oxygen species in the leach matrix. Detection limits calculated for 3s pressure injection at 50 mbar ranged between 0.5-2 microM. The method was linear over the ranges 40-8000, 10-2000, 10-2000, and 5-2000 microM for thiosulfate, trithionate, tetrathionate, and pentathionate, respectively. The technique was applied successfully to leach liquors containing 0.5 M ammonium thiosulfate, 2 M ammonia, 0.05 M copper sulfate and 20% w/v gold ore, diluted 1:100 prior to analysis.

Electrolytes↗
PubMed2005

Thiomicrospira arctica sp. nov. and Thiomicrospira psychrophila sp. nov., psychrophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacteria isolated from marine Arctic sediments.

Two psychrophilic, chemolithoautotrophic, sulfur-oxidizing bacteria were isolated from marine Arctic sediments sampled off the coast of Svalbard with thiosulfate as the electron donor and CO(2) as carbon source. Comparative analysis of 16S rRNA gene sequences suggested that the novel strains, designated SVAL-D(T) and SVAL-E(T), represent members of the genus Thiomicrospira. Further genotypic (DNA-DNA relatedness, DNA G+C content) and phenotypic characterization revealed that the strains represent members of two novel species. Both organisms are obligately autotrophic and strictly aerobic. Nitrate was not used as an electron acceptor. Chemolithoautotrophic growth was observed with thiosulfate, tetrathionate and sulfur. The temperature limits for growth of both strains were between -2 degrees C and 20.8 degrees C, with optima of 11.5-13.2 degrees C (SVAL-E(T)) and 14.6-15.4 degrees C (SVAL-D(T)), which is about 13-15 degrees C lower than the optima of all other recognized Thiomicrospira species. The maximum growth rate on thiosulfate at 14 degrees C was 0.14 h(-1) for strain SVAL-E(T) and 0.2 h(-1) for strain SVAL-D(T). Major fatty acids of SVAL-D(T) are C(16 : 1), C(18 : 0) and C(16 : 0), and those of SVAL-E(T) are C(16 : 1), C(18 : 1), C(16 : 0) and C(14 : 1). Cells of SVAL-D(T) and SVAL-E(T) are rods, like those of their closest relatives. To our knowledge the novel strains are the first psychrophilic, chemolithoautotrophic, sulfur-oxidizing bacteria so far described. The names Thiomicrospira arctica sp. nov. and Thiomicrospira psychrophila sp. nov. are proposed for SVAL-E(T) (=ATCC 700955(T)=DSM 13458(T)) and SVAL-D(T) (=ATCC 700954(T)=DSM 13453(T)), respectively.

Arctic Regions↗
PubMed1991

Use of a Salmonella typhimurium-derived virulence probe in the detection of Salmonella sp. and in the characterization of S. cholerae-suis virulence plasmids.

A genetic probe encoding a virulence gene from Salmonella typhimurium was useful in the detection of Salmonella from feces during an outbreak of salmonellosis at a local dairy. A 3.2-kb BamHI restriction endonuclease fragment of the S. typhimurium virulence plasmid, pStSR100, has been useful as a DNA probe for both detection of Salmonella sp. and characterization of virulence plasmids from numerous field isolates. This virA probe hybridizes to a highly conserved gene carried on the large virulence plasmids of invasive Salmonella isolates. Colony blots prepared from feces directly plated onto MaConkey's agar failed to detect low numbers of Salmonella sp. However, hybridization of the VirA probe to vacuum blots or colony blots prepared from feces in tetrathionate enrichment broth incubated for 16 hours at 37 C was effective for detecting Salmonella sp. and resulted in an 85.9% correlation with culture results. The probe also demonstrated the highly conserved nature (96%) of the virulence gene among S. cholerae-suis isolate plasmids detected using Southern blot analysis.

Animals↗